Complete mitochondrial genome of Lepidocephalichthys berdmorei and its phylogenetic status within the family Cobitidae (Cypriniformes)

#ZooKeys

ZooKeys 1221: 51-69 (2024)
DOI: 10.3897/zookeys.1221.129136

Research Article

Complete mitochondrial genome of Lepidocephalichthys
berdmorei and its phylogenetic status within the family Cobitidae

(Cypriniformes)

Min Zhou', Cheng Wang, Ziyue Xu’, Zhicun Peng’, Yang He’, Ying Wang!2©

1 Hubei Engineering Research Center for Protection and Utilization of Special Biological Resources in the Hanjiang River Basin, Jianghan University, Wuhan, China
2 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China

3 Academy of Plateau Science and Sustainability, Qinghai Normal University, Xining, China

Corresponding author: Ying Wang (xinyuanwangying@163.com)

OPEN Qaccess

Academic editor:

Maria Elina Bichuette
Received: 6 June 2024
Accepted: 28 October 2024
Published: 10 December 2024

ZooBank: https://zoobank.org/
DEE211BF-EF4D-42E2-AB1D-
C82F8F8F4DC5

Citation: Zhou M, Wang C, Xu

Z, Peng Z, He Y, Wang Y (2024)
Complete mitochondrial genome of
Lepidocephalichthys berdmorei and its
phylogenetic status within the family
Cobitidae (Cypriniformes). ZooKeys
1221: 51-69. https://doi.org/10.3897/
zookeys.1221.129136

Copyright: This is an open access article
distributed under the terms of the CCO Public
Domain Dedication.

Abstract

In this study, the complete mitochondrial genome of Lepidocephalichthys berdmorei was
first determined by the primer walking sequence method. The complete mitochondrial
genome was 16,574 bp in length, including 13 protein-coding genes (PCGs), 22 transfer
RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region (D-loop). The
gene arrangement pattern was identical to that of other teleosts. The overall base com-
position was 29.9% A, 28.5% T, 25.5% C, and 16.1% G, with an A+T bias of 58.4%. Further-
more, phylogenetic analyses were conducted based on 13 PCGs from the mitochondrial
genomes of 18 cobitid species using with three different methods (Neighbor-joining,
Maximum likelihood, and Bayesian inference). All methods consistently showed that the
four species of the genus Lepidocephalichthys form a monophyletic group. This study
would provide effective molecular information for the Lepidocephalichthys species as
well as novel genetic marker for the study of species identification.

Key words: Gene arrangement pattern, Lepidocephalichthys berdmorei, mitochondrial
genome, phylogenetic analysis

Introduction

Lepidocephalichthys berdmorei (Blyth 1860)belongs to the genus Lepidoce-
phalichthys within the family Cobitidae, which is widely distributed in the Ir-
rawaddy, Sittang, Salween, Chao Phraya, Mekong basins of Burma, Thailand,
and China (Kottelat and Lim 1993). According to FishBase, there are approx-
imately 25 valid species in the genus Lepidocephalichthys (Froese and Pauly
2024). The lack of reliable morphological characteristics, coupled with the
widespread misapplication of names, has made it challenging to differenti-
ate this species from its close relatives. For instance, the close resemblance
in physical features between L. thermalis and L. berdmorei poses a signifi-
cant challenge in morphological differentiation (Kottelat 2012). Therefore,
molecular information is necessary for an additional method to delimit and
identify species. Lepidocephalichthys berdmorei is a small-sized freshwater

51

Min Zhou et al.: The mitochondrial genome of Lepidocephalichthys berdmorei

fish species, that inhabits hill swift streams, and lakes with sandy and gravel
bottoms (Kamei et al. 2023). In recent years, due to over-exploitation, dam-
age to spawning beds, and construction of the hydroelectric dam in the Lan-
cang River, the wild population size of L. berdmorei has declined dramatically
(Buj et al. 2015; Zhang et al. 2019).

The mitochondrial genome (mtDNA) is a circular double-stranded molecule
consisting of 13 PCGs, 22 tRNAs, two rRNAs, and a control region (D-loop)
(Anderson et al. 1981; Boore 1999; Shen et al. 2020; Chu et al. 2022; Jia et al.
2023). Traditional morphological and biological approaches have focused on
the ecological characteristics of populations and reproduction, with relatively
little molecular research in the genus Lepidocephalichthys (Gohain and Deka
2017; Trif et al. 2022). Because of its limited recombination, highly conserved
gene content, maternal inheritance and moderate evolutionary speed, mtDNA
is now widely used to study population genetics, phylogeny, and species iden-
tification (Avise et al. 1987; Harrison 1989; Boore and Brown 1998; Ballard and
Whitlock 2004; Galtier et al. 2009; Sureandiran et al. 2023). As proof, Wang et
al. (2021) successfully identified fish species from the Xiangjiaba reservoir in
Jinsha River using mitochondrial DNA barcoding. Goswami et al. (2022) char-
acterized the genetic diversity of ten loaches from northeastern India based
on sequence fragments of cox7, cytb, and 16S rRNA genes; Zhang et al. (2023)
demonstrated that the evolutionary position of Rectoris luxiensis (Wu et al.
1977) was consistent with traditional taxonomy through phylogenetic analysis
of mitochondrial genomes. Currently, four mitochondrial genomes have been
reported in NCBI databases, including L. micropogon (Blyth 1860), L. guntea
(Hamilton 1822), L. hasselti (Valenciennes and Cuvier 1846), and L. annanda-
lei (Chaudhuri 1912). Nevertheless, the complete mitochondrial genome of
L. berdmorei has not been reported until now.

In this study, the complete mitochondrial genome of L. berdmorei was se-
quenced for the first time. The variation in tRNA length, position, and size of the
control region, and the codon usage bias were analyzed. Subsequently, the 13
PCGs were concatenated and utilized, with those of other cobitids, to confirm
the phylogenetic position of L. berdmorei. Therefore, these findings will provide
valuable information and contribute to future species comparison and evolu-
tionary research.

Materials and methods
Sample collection and DNA extraction

An adult individual of L. berdmorei was obtained in 2020 from the Mengla
town, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China
(21°57'70"N, 101°60'54"E) (Suppl. material 1: fig. S1). Species were identi-
fied using the original morphological descriptions in the Fauna Sinica field
guides (Chen 1998). After initial morphological identification, the specimen
was deposited in the Animal Genetics Center of Jianghan University under
the voucher number JHU202012029. A 40-50-mg fin clip was collected and
preserved in 95% ethanol at 4 °C. Total genomic DNA was extracted from
caudal fin tissue using the traditional phenol-chloroform method (Sambrook
and Russell 2001).

ZooKeys 1221: 51-69 (2024), DOI: 10.3897/zookeys.1221.129136 52

Min Zhou et al.: The mitochondrial genome of Lepidocephalichthys berdmorei

Mitogenome sequencing, assembly, and annotation

Eight pairs of primers (Suppl. material 1: table S1) were designed based on
the mtDNA sequences of closely allied species. The PCR conditions were as
follows: initial denaturation at 94 °C for 2 min, then 35 cycles of denaturation
at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min,
followed by the final extension at 72 °C for 10 min. All obtained fragments were
quality-proofed and searched via BLAST in the NCBI database to confirm that
the amplicon is the actual target sequence.

Sequences were assembled manually by the Seqman program using DNAs-
tar v. 7.1 software (Burland 2000). The mitochondrial genome was annotated
roughly following the procedure described before (Wang et al. 2011, 2018). The
PCGs, rRNA genes, tRNA genes, and one control region of the mitochondrial ge-
nome were annotated by MitoAnnotator (http://mitofish.aori.u-tokyo.ac.jp/an-
notation/input.html) (Iwasaki et al. 2013). Their secondary structures of tRNAs
were predicted by tRNAScan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/; Lowe
and Eddy 1997) and Forna (force-directed RNA) (Kerpedjiev et al. 2015).

The base composition and relative synonymous codon usage (RSCU) of the
mitogenome were calculated and produced using PhyloSuite v. 1.2.3 (Zhang
et al. 2020) and MAGA X (Kumar et al. 2018). The formulas to calculate the
nucleotide composition of skew are as follows: AT-skew = (A — T)/ (A +T) and
GC-skew = (G — C)/ (G + C) (Perna and Kocher 1995).

Phylogenetic analyses

To verify the phylogenetic position of L. berdmorei, 17 mitogenome sequences
from GenBank were retrieved (Suppl. material 1: table S2; Saitoh et al. 2006,
2010). The 13 PCGs for each species were concatenated and then aligned by
program MAFFT using default settings (Katoh et al. 2002), and phylogenetic
analyses were performed using Neighbor-joining (NJ), Maximum likelihood
(ML), and Bayesian inference (BI) methods. To root the phylogenetic tree, Syn-
crossus beauforti (Smith 1931)and S. hymenophysa (Bleeker 1852) from Botii-
dae were chosen as outgroups.

A NJ phylogenetic tree was constructed using MEGA 7 (Kumar et al. 2016)
with 1,000 bootstrap replicates. The ML method was assembled in RAXML
7.0.3 (Stamatakis 2006), with 1,000 bootstrap replicates. GTR + F +1 + G4 was
selected as best-fit model according to Bayesian Information Criterions (BIC)
estimated by ModelFinder (Kalyaanamoorthy et al. 2017). The BI phylogeny
was carried out using MrBayes v. 3.2.7a (Ronquist et al. 2012) under the best-
fit models with 5,000,000 generations in two runs of eight chains each.

Abbreviations

Mitogenome, mitochondrial genome; mtDNA, mitochondrial DNA; PCGs, pro-
tein-coding genes; tRNA, transfer RNA; rRNA, ribosomal RNA; atp6 and atp8,
ATPase 6 and ATPase 8; cox1-3, cytochrome oxydasec subunits I-Ill; cytb,
cytochrome b; LA-PCR, long and accurate polymerase chain reaction; nd1-6,
NADH dehydrogenase subunits 1-6; nd4/, NADH dehydrogenase subunits 4L;
A+tT, A+T rich region; RSCU, relative synonymous codon usage; trnA, tRNA;

ZooKeys 1221: 51-69 (2024), DOI: 10.3897/zookeys.1221.129136 53

Min Zhou et al.: The mitochondrial genome of Lepidocephalichthys berdmorei

trnC, tRNA“: trnD, tRNA“: trnE, tRNA&; trnF, tRNA”’®; rrnS, 72S rRNA; rrnL, 76S
rRNA; trnG, tRNA®; trnH, tRNAs; trnl, tRNA"®; trnK, tRNA”*; trnL1, tRNA“):
trnL2, tRNA“°““\4°). trnM, tRNA: trnN, tRNA2*: trnP, tRNA’: trnQ, tRNA®": trnR,
tRNA‘9; trnS1, tRNA‘): trnS2, tRNASe(6°)- trnT, tRNA™: trnV, tRNA. trnW,
tRNA” trnY, tRNA™: DHU, Dihydrouracil; NJ, Neighbor-joining; ML, Maximum
likelihood; BI, Bayesian inference.

Results and discussion
Mitogenome organization and nucleotide composition

The length of the complete mitochondrial genome of L. berdmorei is
16,574 bp (GenBank accession number: OP651767). The complete mitochon-
drial genome of L. berdmorei shares high similarity in gene arrangement, base
composition, and codon usage pattern with those of other teleosts, indicating
that the mitochondrial genome is highly conserved in evolution (Boore 1999;
Taanman 1999; Broughton et al. 2001; Zou et al. 2019; Shen et al. 2020; Wang
et al. 2020; Yu et al. 2021). The mitogenome is a circular double-stranded mol-
ecule with a highly conserved structure, consisting of 13 PCGs, 22 tRNA genes,
two rRNA genes, and a control region (D-loop) (Fig. 1, Table 1).

The overall base composition is 29.9% for A, 16.1% for G, 25.5% for C, and
28.5% for T, which is consistent with the lowest frequency for G among the four
bases in fish mitochondrial genomes, and revealing the A+ T-rich content (58.4%)
(Mayfield and McKenna 1978; Meyer 1993). Based on the analysis of nucleotide
composition, this complete sequence exhibits a clear bias towards A and T (AT-
skew = 0.02, GC-skew = -0.23) (Suppl. material 1: table S3). Both L. berdmorei
and 58 species of Cobitidae exhibit an AT bias in their mitogenomes, but the
A+T-rich content size varied among species, and it may be related to factors
such as natural mutations and selection pressures during replication and tran-
scription (Zhong et al. 2002; Yu et al. 2021). Hence, during the processes of rep-
lication and transcription, the asymmetry in nucleotide composition was used
to infer the direction of gene orientation and replication (Francino and Ochman
1997; Frank and Lobry 1999; Satoh et al. 2016; Moeckel et al. 2023).

Overlaps and non-coding intergenic spacers

Cobitidae mitogenomes range from 16,574 bp (L. berdmorei) to 16,646 bp (Co-
bitis striata (Ikeda, 1936)) in length (Suppl. material 1: table S2). With a few
exceptions, the gene arrangements of fish mitogenomes are usually conserved
(Anderson et al. 1981; Chang et al. 1994; Satoh et al. 2016; Chu et al. 2022). A
typical feature in the mitochondrial genome of teleosts is the overlap of nucle-
otides between adjacent genes, suggesting that the size of mitochondrial DNA
is very compact and economical, with potential kinetic advantages during the
process of replication (Boore 1999; Curole and Kocher 1999; Taanman 1999;
Wang et al. 2011; Satoh et al. 2016; Zou et al. 2017; Zou et al. 2018; Zhang et
al. 2023). Similarly, in the L. berdmorei mitochondrial genome, there are over-
laps and intervals of different lengths in all genes except for trnF/rrnS, rrnS /
trnV, trnV/rrnL, rrnL/trnL2, trnM/nd2, trnC/trny, cox2/trnK, trnG/nd3, trnR/nd4l,
trnH/trnS2, trnL2/nd5, nd6/trnE, and cytb/trnT. They have the longest spacer in

ZooKeys 1221: 51-69 (2024), DOI: 10.3897/zookeys.1221.129136 54

Min Zhou et al.: The mitochondrial genome of Lepidocephalichthys berdmorei

trnN/trnC (30 bp) and the largest genetic overlap in atp8/atp6 (10 bp) (Table 1).
The length of the mitochondrial genome is related to the various overlaps and
intergenic spacers between adjacent genes (Huang and Liu 2010). Interesting-
ly, the presence of a specific 3 bp insertion (GCA) in the overlapping atp8—atp6
motif of both L. berdmorei and other loaches compared to the conserved motif
of 7 bp (ATGATAA) in other Cypriniformes fishes, suggests that this insertion
is characteristic of loaches (Kanu et al. 2016; Wu et al. 2016; Yu et al. 2016; Yu
et al. 2021). They may influence the expression of neighboring genes, regulate
the normal operation of mitochondrial function, and participate in the process
of mitochondrial genome replication and transmission (Boore 1999; Taanman
1999: D’Souza and Minczuk 2018).

™ —tRNA-Phe

4

ig

2

eu

2
I

Magy.
“<{Yy,.

—t NA-\Wle
—4kb . TRNA-Met

Lepidocephalichthys berdmorei

16,574 bp

TOS

Figure 1. Gene map and organization of the mitochondrial genome of Lepidocephalichthys berdmorei. Photograph of
L. berdmorei from https://fishbase.se/summary/Lepidocephalichthys-berdmorei.html.

ZooKeys 1221: 51-69 (2024), DOI: 10.3897/zookeys.1221.129136 55

Min Zhou et al.: The mitochondrial genome of Lepidocephalichthys berdmorei

Table 1. Organization of the mitochondrial genome of Lepidocephalichthys berdmorei.

Locus

tRNA?*(S)

12S rRNA
tRNAY2'(V)

16S rRNA
tRNA“<“\744)(L 7)
nd1

tRNA4"(1)
tRNA®'"(Q)
tRNA™*(M)
nd2
tRNA™(W)
tRNA4"(A)
tRNA*="(N)
tRNA%S(C)
tRNA™'(Y)
Cox1
tRNAS*764)(S7)
tRNA**(D)
COx2
tRNAS(k)
atp8

atp6

Cox3
tRNA®(G)

nd3

tRNA*9(R)
nd4l

nd4

tRNA"'s(H)
tRNAS@"6CT) ($2)
tRNA““\749)(L 2)
nd5

nd6

tRNA®"(E)

cytb

tRNA™'(T)
tRNA??(P)
D-loop

Position 7 ; : Codon f
Size (bp) | Intergenic nucleotides? Anti-codon Strand?

From To Start Stop
1 69 69 0 = = GAA H
70 1019 950 0 = = = H
1020 1091 72 0 - = TAC H
1092 2/67 1676 0 = = = H
2768 2842 iO 1 = = TAA H
2844 3818 975 5 ATG TAA 7 H
3824 3895 72 is — 7m GAT H
3894 3964 ra 1 = = TTG L
3966 4034 69 0 = = CAT H
4035 5081 1047 <2 ATG TAG - H
5080 5148 69 2 = = TCA H
5151 5219 69 1 = iz TGC L
5221 9293 73 30 = = GTT L.
5324 5390 67 0 = =; GCA L
5391 5459 69 1 z = GTA E
5461 7011 1551 2 GTG TAA = H
7014 7084 Fl 1 - i TGA L
7086 7158 73 13 = = GTC H
7172 7862 691 0 ATG T z H
7863 7938 76 1 G = TTT H
7940 8107 168 -10 ATG TAA = H
8098 8781 684 -1 ATG TAA = H
8781 9566 786 il ATG TAA 7 H
9566 9638 73 0 = a TG H
9639 9989 351 ae ATG TAG = H
9988 10056 69 0 = = TCG H
10057 10353 297 -/ ATG TAA 4 H
10347 11729 1380 =] ATG TAG * H
11729 11797 69 0 = iz GTG H
11798 11866 69 1 = ? GCT H
11868 11940 Wess 0 = = TAG H
11941 13779 1839 -4 ATG TAA a H
13776 14297 522 0 ATG TAA = L
14298 14366 69 5 - - TLTG L
14372 Th5i2 1141 0 ATG Ti = H
15513 15584 72 -2 = = TGT H
15583 15652 70 0 = = TGG L
15653 16574 922 0 = = = H

@ Negative value indicates the overlapping sequences between adjacent genes.
> H: heavy strand; L: light strand.

PCGs and codon usage

The length of PCGs was 11,413 bp (68.86%) and it blanketed 7 NADH dehydro-
genases (nd1-6 and nd4I/), three cytochrome coxidases (cox7—3), two ATPases
(atp6 and atp8) and one cytochrome b (cytb). The size of PCGs ranged from
nd4I (297 bp) to nd5 (1839 bp). As in other vertebrates, the nd6 and eight tRNA
genes (tRNA®", tRNA‘, tRNA4", tRNA%S, tRNA”, tRNAS*, tRNAP?, and tRNAS")
are encoded on the light strand, and the others are encoded on the heavy strand
(Fig. 1, Table 1) (Wen et al. 2017; Zou et al. 2017; Yu et al. 2021). In addition, the

ZooKeys 1221: 51-69 (2024), DOI: 10.3897/zookeys.1221.129136 56

Min Zhou et al.: The mitochondrial genome of Lepidocephalichthys berdmorei

bias of nucleotide composition was estimated (Suppl. material 1: table S3). All
13 PCGs showed a significant negative GC-skew. It may be that mutations in
the replication process or adaptive evolution cause GC-skew. However, how to
explain this unusual GC-skew needs further study.

Further analysis revealed that among 13 PCGs, most mitochondrial genes of
L. berdmorei started with codon ATG, while only the cox7 gene began with codon
GTG. Unconventional start codons are a common phenomenon within the mitog-
enomes of fish (Zhang and Shen 2019; Yu et al. 2021). Eight of the PCGs are end-
ed by TAA termination codons. The nd2, nd3, and nd4 genes ended with TAG stop
codons. The cox2 and cytb use incomplete stop codons (T-) (Table 1). The rela-
tive synonymous codon usage (RSCU) denotes the differential usage of synon-
ymous codons encoding the same amino acid. Essentially, the RSCU value was
calculated by dividing the amino acids encoded by the same codons and their
probability of appearing in the same codons (Sharp and Li 1986). The RSCUs of
L. berdmorei mitogenome (Fig. 2, Table 2) show a clear preference for the usage
of A and T. The total number of codons in the L. berdmorei mitochondrial genome
is 5,524. After excluding the four stop codons (UAA(*), UAG(*), AGA(*), AGG(*)),
among the 64 codons, 31 codons have an RSCU value greater than 1, indicating
that these codons are prioritized more highly. For instance, six codons (UUA(L),
UUG(L), CUU(L), CUC(L), CUA(L), CUG(L)) coded for leucine with preference for
UUA. RSCU values for these six codons were 1.68, 0.64, 1.47, 0.65, 0.99 and 0.56,
respectively. The most commonly used codon is UUU-Phe (F), followed by UUA-
Leu2 (L), AAA-Lys (K), and AUU-Ile (I). The least used amino acids are Ala (GCG)
and Arg (CGU). Our results show that the codon distribution is largely consistent
with the mitogenomes of Cobitinae studied previously (Yu et al. 2021).

RSCU

Ala Arg Asn Asp Cys Gln Glu Gly His [le Leul Leu2 Lys Met Phe Pro Serl Ser2 Thr Trp Tyr Val

Figure 2. The relative synonymous codon usage (RSCU) of L. berdmorei mitogenome.

ZooKeys 1221: 51-69 (2024), DOI: 10.3897/zookeys.1221.129136 57

Min Zhou et al.: The mitochondrial genome of Lepidocephalichthys berdmorei

Table 2. Codon usage in the mitochondrial genome of Lepidocephalichthys berdmorei.

Codon
UUU(F)
UUC(F)
UUA(L)
UUG(L)
CUU(L)
CUC(L)
CUA(L)
CUG(L)
AUU(I)
AUC(I)
AUA(M)
AUG(M)
GUU(V)
GUC(V)
GUA(V)
GUG(V)

Count
201
113
188

71
164
73
111
63
170
98
98

RSCU
1.28
0.72
1.68
0.64
1.47
0.65
0.99
0.56
127
0.73
1.18
0.82

1.6
0.57
i Rai
0.61

Codon Count RSCU Codon Count RSCU Codon Count RSCU

UCU(S) | 103 1.37. | UAU(Y) | 118 1.19 | UGU(C) 40 0.95
ucc(s) | 110 1.46 UAC(Y) 80 0.81 | UGC(C) 44 1.05
UCA(S) 68 0.9 UAA(*) 156 1.66 | UGA(W) 75 1.18
UCG(S) 40 0.53 UAG(*) 99 1.06 | UGG(W) 52 0.82
CCU(P) | 110 1.01 | CAU(H) 95 0.95 | CGU(R) 21 0.65
ccc(P) | 134 1.23 | CAC(H) | 104 1.05 | CGC(R) 37 1.15
CCA(P) | 137 1.26 CAA(Q) | 156 1.34 | CGA(R) 49 1.52
CCG(P) 55 0.5 | CAG(Q) 76 0.66 | CGG(R) 22 0.68
ACU(T) | 102 1.06 AAU(N) 134 1.06 | AGU(S) 49 0.65
ACC(T) | 120 1.25 AAC(N) | 118 0.94 AGC(S) 82 1.09
ACA(T) | 128 1.33. AAA(K) | 172 1.36 | AGA(*) 75 0.8
ACG(T) 35 0.36 AAG(K) 81 0.64 AGG(*) 45 0.48
GCU(A) 82 1.13. | GAU(D) 63 0.92 | GGU(G) 44 0.81
ecc(ay | 1494 1.57. | GAC(D) 74 1.08 | GGC(G) 53 0.98
GCA(A) 75 1.03 | GAA(E) 83 1.06 | GGA(G) 70 1.3
GCG(A) 20 0.27. | GAG(E) 74 0.94 GGG(G) 49 0.91

Transfer and ribosomal RNA genes

The complete mitogenome of L. berdmorei contains 22 tRNA genes with a size
of 1,559 bp, 14 of which are located on the H-strand while the others are on the
L-strand (Table 1). The 22 tRNA genes range from 67 bp to 76 bp in length, of
which the shortest was tRNA“ (67 bp) and the longest was tRNA (76 bp). The
color in Fig. 3 represents the type of tRNA structure in which the nucleotide is
located. All tRNA genes have a typical cloverleaf secondary structure except
tRNAS*6°) lacking the Dihydrouracil (DHU) stem (Fig. 3). It is a common feature
in many mitogenomes of metazoans, and can be integrated into ribosomes by
adjusting its structure and function to fulfil its function of carrying and trans-
locating amino acids (Watanabe et al. 2014; Liu et al. 2021; Xing et al. 2022).

The most prevalent non-Watson-Crick base pairs in the secondary structure
of tRNAs are A-C (e.g., trnl, trnH, trnM, trnV, trnS1, trnT, trnW, and trnF), followed
by T-T (trnQ and trnN), which are mostly located in the DHU, anticodon stems
and acceptor (Fig. 3). And these mismatches may be modified by post-tran-
scriptional editing processes without causing amino acid transport disorders
(Tomita et al. 1996).

The lengths of 72S rRNA and 16S rRNA genes were 950 bp and 1,676 bp,which
are located on the H strand (Table 1). They are bordered by tRNA’? and tR-
NA‘eA44) and separated by tRNA”’. Both the lengths and base compositions
of 72S rRNA and 16S rRNA are almost identical among the reported Cobitidae
fishes (Kottelat and Lim 1993; Nalbant 1993; Yu et al. 2016; Shen et al. 2020;
Chu et al. 2022; Ke et al. 2023). It shows a positive AT-skew (0.23) and a neg-
ative GC-skew (-0.07) (Suppl. material 1: table S3). Compared to entire mito-
chondrial genome, the 76S rRNA is a non-coding gene that evolves slowly, and
it contains sufficient number of polymorphisms to distinguish species (Lakra et
al. 2009; Sarri et al. 2014; Hossain et al. 2019). The 12S rRNA is also frequently
considered as a DNA meta barcoding in fish identification and phylogenetic
studies (Miya et al. 2015).

ZooKeys 1221: 51-69 (2024), DOI: 10.3897/zookeys.1221.129136 58

Min Zhou et al.: The mitochondrial genome of Lepidocephalichthys berdmorei

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Figure 3. Putative secondary structure of tRNAs. Stems (typically helical) are shown in green, multiple loops (junctions)
are shown in red, interior loops are shown in yellow, hairpin loops are shown in blue, and 5’ and 3’ unpaired regions are

shown in orange.

ZooKeys 1221: 51-69 (2024), DOI: 10.3897/zookeys.1221.129136

Control region

The only large control region of L. berdmorei mitogenome is the D-loop, located
between the tRNA’ and tRNA™®, with a length of 922 bp (Fig. 1, Table 1). It plays
a role in the regulation of replication and transcription and is the most rapid-
ly evolving and changing region of the mitochondrial genome (Clayton 1982,
1991; Shadel and Clayton 1997; Zhou et al. 2014; Gao et al. 2023). The A+ T-rich
content of the L. berdmorei D-loop region is 66.27%, which is higher than the av-
erage value of the whole mitogenome (58.43%) and 13 PCGs (56.11-61.07%)
(Suppl. material 1: table S3), as found in other vertebrates (Brown et al. 1986;
Saccone et al. 1987; Zou et al. 2017; Ke et al. 2023).

59

Min Zhou et al.: The mitochondrial genome of Lepidocephalichthys berdmorei

In addition to gene duplication and insertion/deletion events, the main cause
of mitochondrial genome size variation is differences in control region length
(Mignotte et al. 1990; Lee et al. 1995; Pereira 2000; Minhas et al. 2023). Previ-
ous studies have demonstrated that tandem repeat sequences are prevalent
in the D-loop of teleost lineage (Lee et al. 1995; Nicholls and Minczuk 2014;
Jemt et al. 2015; Xu et al. 2016; Ke et al. 2023). It is worth noting that the copy
number not only varies between species, but also among individuals within the
same species (Norman et al. 1994; Lunt et al. 1998; Boore 1999; Xu et al. 2021).
Thus, compared with the complex and large eukaryotic genome, the mitochon-
drial genome is simple in structure with shorter sequences, contains both con-
served and highly variable regions, and can be used for taxonomic identifica-
tion of species at different levels of evolution (Pereira et al. 2008; Jamandre et
al. 2014; Nicholls and Minczuk 2014; Jemt et al. 2015; D'Souza and Minczuk
2018). Nevertheless, multiple duplicate regions have been found in some spe-
cies that may adversely affect PCR amplification, sequencing, or both (Singh et
al. 2008; Cadahia et al. 2009). As a result, researchers have avoided using this
region for phylogenetic purposes, focusing instead on rRNA or PCGs (Slech-
tova et al. 2008; Wang et al. 2021; Sureandiran et al. 2023; Zhang et al. 2023).

Phylogenetic analysis

Cobitidae belongs to Osteichthyes, Cyprinidformes, and has three subfamilies: No-
emacheilinae, Botiinae and Cobitinae (Hora 1932; Nalbant 1993; Tang et al. 2005;
Slechtova et al. 2008; Chu et al. 2022). Sawada (1982) proposed a phylogeny of
the Cobitoidea (limited to loaches) as (Botiinae + Cobitinae) + (Nemacheilinae +
Homalopterinae) based on 52 osteological characters. Nevertheless, due to their
morphological similarity and frequent overlap, differentiating species within Cobiti-
dae based solely on morphology is a challenging endeavor (Kottelat and Lim 1993;
Nalbant 1993; Shen et al. 2020; Ke et al. 2023). In order to determine the phyloge-
netic status of L. berdmorei in the family Cobitidae, 17 complete mitochondrial
genomes from the GenBank database were selected to reconstruct phylogenetic
trees. Based on the 13 PSGs concatenated dataset, the NJ, ML and BI phylogenies
generated identical topology with high bootstrap support and posterior probability
values, respectively (Fig. 4). All trees presented two major clades corresponding to
the outgroup. Canthophrys is located at the base of the phylogenetic tree. Our re-
sults are generally consistent with the traditional morphological classification and
recent molecular studies (Hora 1932; Slechtova et al. 2008; Sudasinghe et al. 2024).

Firstly, the phylogenetic tree revealed that L. guntea, L. hasselti, and L. berd-
morei clustered as a monophyletic clade, followed by a clade with L. micropo-
gon with high bootstrap support. Secondly, the genus Lepidocephalichthys and
Pangio which formed a sister branch with high bootstrap support and posterior
probability values, which was consistent with the previous study (Slechtova et
al. 2008; Yu et al. 2021). Notably, Slechtova et al. (2008) found that the gen-
era Lepidocephalichthys and Pangio were considered as a sister group in the
RAG-1 phylogeny; but this relationship was not supported by the cytb dataset.
Meanwhile, based on cyt b and RAG-1 datasets, these four genera of Cobitidae
(Cobitis, Niwaella, Misgurnus, and Koreocobitis) form a distinct monophyletic
group (Slechtova et al. 2008). Generally, from the phylogenetic tree of genetic
evolution, the evolutionary status of L. berdmorei was defined.

ZooKeys 1221: 51-69 (2024), DOI: 10.3897/zookeys.1221.129136 60

Min Zhou et al.: The mitochondrial genome of Lepidocephalichthys berdmorei

100/100/1.00 Koreocobitis rotundicaudata
100/100/1.00 Koreocobitis naktongensis
100/100/1.00 Misgurnus nikolskyi
100/100/1.00 Misgurnus mizolepis
100/100/1.00 Niwaella delicata (1)
100/100/1.00 Niwaella delicata (2) Cobitidae
100/100/1.00 Cobitis lutheri
100/76/0.99 Cobitis striata
100/92/1.00 Lepidocephalichthys guntea
100/99/1.00 Lepidocephalichthys hasselti
pes Lepidocephalichthys berdmorei
Sane 100/80/1.00 Lepidocephalichthys micropogon
100/100/1.00 Pangio oblonga
Pangio cuneovirgata
100/100/1.00 Canthophrys gongota (1)
Canthophrys gongota (2)
100/100/1.00 Syncrossus beauforti

Outgroup

Syncrossus hymenophysa

Figure 4. Phylogenetic tree of Cobitidae and two outgroups based on the NJ, ML and BI analysis of 13 concatenated
protein-coding genes. Tree topologies produced by NJ, ML methods, and BI analysis were equivalent. The numbers at the
nodes represent bootstrap support values for NJ and ML analyses and Bayesian posterior probability, sequentially, and
the red branch represents the specie in this study.

Conclusions

In conclusion, the complete mitochondrial DNA sequence of L. berdmorei is de-
termined for the first time by the primer walking sequence method. The mitog-
enome is 16,574 bp in length, and encodes all of the 37 genes that are typical
for Cobitidae fish. We compared mtDNA from L. berdmorei with that of other te-
leost and analyzed mitogenome composition, PCGs, and codon usage, transfer
and ribosomal RNA genes, and noncoding regions (control region, intergenic
spacers). The generated phylogenetic trees yielded convincing evidence that
the genus Lepidocephalichthys formed a monophyletic group. These findings
will provide new insights into better understanding the phylogenetic status of
this intriguing and ecologically important group.

Acknowledgements

This work was supported by grants from the National Natural Science Founda-
tion of China (31702016), and Jianghan University Research Project Funding
Plan (2023KJZX42). We greatly appreciate the valuable suggestions of two
anonymous referees.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

All animal protocols have been reviewed and approved by the experimental animal wel-
fare and ethics review committee of Jianghan University, Qinghai Normal University, and
Chinese Academy of Sciences.

ZooKeys 1221: 51-69 (2024), DOI: 10.3897/zookeys.1221.129136 61

Min Zhou et al.: The mitochondrial genome of Lepidocephalichthys berdmorei

Funding

This work was supported by the National Natural Science Foundation of China (Grant
number 31702016) and Jianghan University Research Project Funding Plan (Grant num-
ber 2023KJZX42).

Author contributions

Ying Wang contributed to the concept and design of the study. Sample collection and
preparation of materials were done by Cheng Wang and Zhicun Peng. Ziyue Xu, Yang He
and Min Zhou performed the data analysis and interpretation, and Min Zhou wrote the
first draft of the manuscript. Ying Wang revised this manuscript. All authors read and re-
vised the manuscript and approved the final version. All authors agree to be accountable
for all aspects of the manuscript.

Author ORCIDs
Ying Wang ® https://orcid.org/0000-0002-8222-7510

Data availability

Genome sequence data that support the findings of this study are openly available from
the GenBank at https://www.ncbi.nlm.nih.gov/, under accession No. OP651767.

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Supplementary material 1
Supplementary files

Authors: Min Zhou, Cheng Wang, Ziyue Xu, Zhicun Peng, Yang He, Ying Wang

Data type: docx

Explanation note: figure S1. Images of biological sample for this study. table $1. Prim-
ers used for amplification of the mitochondrial genome of Lepidocephalichthys berd-
morei. table S2. Species and GenBank accession numbers of mitogenomes used in
this study. table S3. Nucleotide contents of genes and the mitochondrial genome
skew of Lepidocephalichthys berdmorei.

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: https://doi.org/10.3897/zookeys.1221.129136.suppl1

ZooKeys 1221: 51-69 (2024), DOI: 10.3897/zookeys.1221.129136 69